Immunoblotting and immunoelectron microscopy of isolated exosomes. Platelets were incubated with 1 U/mL thrombin for 120 seconds. Following termination, platelets were removed by centrifugation at 800g. Further centrifugation of the supernatant removed the microvesicles. Exosomes were isolated by differential centrifugation through a sucrose gradient. Fractions were collected from the top, and immunoblotting was carried out in each fraction using anti-PrPC Ab 308. The blots were subjected to densitometry and are expressed as mean plus or minus standard error of the mean; n = 3 (A). Fractions 3 and 4 from the sucrose gradient were adsorbed onto formvar-coated grids and double labeled with anti-PrPC Ab 308 followed by an anti-CD62 Ab (D541). The respective secondary Abs were conjugated to 5 nm (anti-PrPC; black arrows) and 10 nm (anti-CD62; white arrows) gold (B-C).
